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With advancements in analytical technologies, such as next-generation sequencing, the industry is transitioning towards next-gen technologies for adventitious agent testing.
In an industry where product recalls and patient litigation can fundamentally undermine profit, adventitious agent testing is crucial in making certain that biologics products are free from contaminants. Consequently, BioPharm International® spoke with Kerstin Brack, PhD, scientific director of Global Biosafety at Charles River Laboratories, to update readers on the state of the industry. Topics of discussion include novel innovations in adventitious agent testing, key ongoing challenges, and what the future of the space may look like.
BioPharm: What are some novel technologies/processes in the adventitious agent testing field? What makes them unique relative to existing procedures?
Brack (Charles River): Traditional test methods, which are used for adventitious agent testing, require the replication of an infectious agent either in culture media, cell cultures, or animals. After a suitable incubation period, the test system is checked for signs of an infection. Such endpoints could be the formation of bacterial colonies, cell death, or disease symptoms in animals. These assays are lengthy and usually take two to four weeks, or even longer, from initiation to read-out.
A major limitation of these assays is that false negative results are obtained when the used test system is not suitable to support the growth of a contaminant present in the tested sample. Conversely, in case of a positive result, further analyses are required to identify the causative agent. In addition to these unspecific screening assays with a broad detection range, polymerase chain reactions (PCRs) are also an important part of the testing strategy which allow the sensitive detection of nucleic acid molecules of specified adventitious agents within one day. However, prior knowledge of the target is needed, and contaminants will not be detected if primers and probes do not bind to their target sequences. If a positive result is obtained, follow-up investigations are needed to determine if the nucleic acid is associated with an infectious agent or if it is non-infectious.
New advanced detection methods have been developed in the past two decades which are suitable to overcome some of the limitations of the traditional testing methods. Most important to mention is next-generation sequencing (NGS) which allows the sensitive detection and simultaneous identification of contaminants within days by unbiased amplification of all present nucleic acid molecules in a sample and high-throughput sequencing. After data processing and bioinformatic analysis, a database is used to map and identify the detected sequences. This technology has the potential to replace the animal- and cell-based adventitious virus tests as well as PCR tests and microbial detection assays. Other examples for novel technologies are rapid sterility tests, which have a reduced incubation period from two weeks to less than one week by using endpoints such as adenosine triphosphate (ATP)-dependent bioluminescence or carbon dioxide-induced pH shift of culture media. These surrogate markers for microbial growth allow an earlier read out and faster time to result compared to the traditional culture methods. In addition, rapid microbiological methods often require less sample volume and have the advantage of being automated and adapted to high-throughput testing of samples.
Other examples for novel technologies are rapid sterility tests, which have a reduced incubation period from two weeks to less than one week by using endpoints such as adenosine triphosphate (ATP)-dependent bioluminescence or carbon dioxide-induced pH shift of culture media. These surrogate markers for microbial growth allow an earlier read out and faster time to result compared to the traditional culture methods. Rapid microbiological methods often require less sample volume and have the advantage [of being] automated and adapted to high-throughput testing of samples.
BioPharm: What are the biggest challenges in adventitious agent testing? What solutions, if any, are in the works to resolve them?
Brack (Charles River): It is a big challenge that traditional test methods have their limitations, as mentioned above, and therefore there is still a risk that adventitious agents are not detected. Furthermore, sample matrices can be toxic for the cells used as detector cells in the in-vitro adventitious virus tests. The cells die after they were exposed to the sample, and an evaluation of the test is not possible. As a consequence, such cytotoxic samples must often be diluted for testing, which lowers the sensitivity of the method. Using NGS for adventitious virus testing can be an alternative for toxic samples matrices. Similarly, live virus vaccine harvests contain the infectious vaccine virus which must be neutralized by specific antibodies prior to adventitious virus tests in cell cultures or animals. If antibodies are not available, NGS is an alternative for adventitious virus detection. The traditional adventitious agent tests are tedious. The in-vitro adventitious virus test, which is routinely used for adventitious virus detection, takes 14 or 28 days, respectively. These timelines are not tolerable for the production of cell therapy products with a short shelf life of only a few days.
BioPharm: Would you characterize adventitious agent testing as a field that has frequent change, or are the technologies/processes relatively constant year-over-year? Why?
Brack (Charles River): Although new analytical test methods for adventitious agent testing have advantages, it takes time for these newer methods to be accepted by the industry and regulating agencies and to be implemented into corresponding guidelines. The current revision of the ICH [International Council for Harmonisation] Q5A guideline (1), 14 years after the last changes in 1999, was driven by the necessity to provide guidance to ensure viral safety of new advanced modalities and new manufacturing processes. Furthermore, next-generation sequencing and broad-range molecular virus detection methods are available today and can be used to supplement or replace traditional virus detection methods. Their integration into the testing strategies also supports the efforts to reduce animal testing in the manufacture of biological products. Thus, multiple significant changes in the field have accumulated and made it necessary to revise the underlying guideline.
BioPharm: Which production stage is most at risk of contamination?
Brack (Charles River): The fermentation phase offers the chance for adventitious agents to enter the manufacturing process, for example via contaminated raw materials. The unprocessed fermenter harvest represents the most relevant process intermediate for adventitious agent testing in batch production. At this stage, potentially present adventitious agents had the chance to accumulate during the fermentation process and are not yet removed or inactivated by the following downstream process. Therefore, unprocessed bulk harvest samples offer the highest probability to detect adventitious agents, if present.
BioPharm: Cell therapies are a bit of a different beast in terms of viral
testing. What unique challenges exist in this space?
Brack (Charles River): Viral testing is of major importance for cell therapy production processes for several reasons. Human cells are susceptible to human virus infections. Additionally, biological raw materials are frequently used for production. On the other hand, there are generally no process steps that are capable of inactivating or eliminating adventitious agents. At the same time, cell therapies often have a reduced shelf life, and test results are required in a short time. Therefore, lengthy traditional test methods are not suitable for viral safety testing of cell therapies, and rapid molecular and microbiological methods are used to address the identified contamination risks.
For cell therapy products, it is therefore critical to ensure the virus safety of the product by testing the used raw materials and starting materials after a thorough risk assessment of the manufacturing process. That said, another challenge is the limited availability of sample material for testing because of small production lots.
BioPharm: Do you have any predictions for how the industry will handle virology testing 10 years from now?
Brack (Charles River): There are published data available which demonstrate that NGS is suitable to replace the traditional in-vivo adventitious virus tests (2). Together with the current efforts to reduce the use of animals for testing, it can be expected that in the coming years animal-based adventitious virus tests will only be used in well-justified exceptional cases.
BioPharm: Do you have any final thoughts on this topic that you’d like to share with our readers?
Brack (Charles River): Technologies for adventitious agent detection will further evolve towards more sensitive and rapid methods with broad detection ranges. Applications based on artificial intelligence and machine learning have the potential to revolutionize this field. However, the development of new technologies takes time. They must be validated thoroughly to demonstrate their suitability before they are accepted by the industry and regulating agencies and are implemented into testing strategies for adventitious agent control in biomanufacturing.
1. ICH, Q5A(R2) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, Step 2b version (2022).
2. Beurdeley-Fehlbaum, P; Pennington, M; Hégerlé, N.; et al., Evaluation of a Viral Transcriptome Next Generation Sequencing Assay as an Alternative to Animal Assays for Viral Safety Testing of Cell Substrates. Vaccine 2023, 41 5383-5391. https://doi.org/10.1016/j.vaccine.2023.07.019
Grant Playter was formerly the associate editor for BioPharm International.